Methylation-associated silencing of tumor suppressor genes is a common event implicated in the formation and progression of cancer. Although the causes of methylation-associated tumor suppressor gene silencing are unknown, it is thought that stable repression of methylated tumor suppressor gene promoters is mediated by one or more methyl-binding proteins (MBPs). These proteins bind specifically to methylated CpG (mCpG) dinucleotides. This proposal addresses the possibility that MBPs play a causal role in tumor suppressor gene inactivation prior to promoter methylation. Specifically, I propose that altered methylation patterns in tumor cells cause the mistargeting of MBPs to methylated regions flanking unmethylated CpG islands. Promoters that are normally protected from inactivation are unable to resist the increased repressor activity from MBPs bound nearby, become silenced and subsequently methylated. The experiments described here will directly test the predictions of this model using the well-characterized mouse adenine phosphoribosyltransferase gene (Aprt) as a model. The objectives of this proposal are: 1) to determine if mistargeting of MBPs via overexpression can cause the aberrant silencing of the endogenous Aprt gene; 2) to determine if unmethylated promoters that are resistant to methylation- associated silencing are also resistant to silencing by MBPs bound upstream; 3) to determine if silencing of an unmethylated promoter by MBPs leads to its methylation. The proposed studies will provide important insights into the nature of aberrant gene silencing in cancer and will reveal functional differences between MBPs.